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in workers and virgin queens of Melipona scutellaris
Douglas Elias Santos, Edmilson Amaral de Souza, Carlos Ueira Vieira, José
Cola Zanuncio, José Eduardo Serrão
To cite this version:
Morphology of mandibular and intramandibular glands
Douglas Elias SANTOS1,Edmilson Amaral de SOUZA2,Carlos Ueira VIEIRA3,
José Cola ZANUNCIO4,José Eduardo SERRÃO1
1Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil 2Departamento de Biologia, Universidade Federal de Viçosa, campus Rio Paranaíba, Rio Paranaíba, MG, Brazil
3Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, 38400-902, Uberlândia, Brazil 4
Departamento de Biologia Animal, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil Received 16 December 2013– Revised 20 May 2014 – Accepted 4 June 2014
Abstract– The interactions of insect societies are associated with chemical signals released by the exocrine glands that control some insect behaviors. Exocrine glands can be classified into different classes: class I release substances that cross the body cuticle and class III glands that have a conducting canal. This study compares the morphology of intramandibular and mandibular glands in virgin queens of different ages, as well as in nurses and forager workers of Melipona scutellaris. We have found the occurrence of two types of glands inside the mandible; glands of class I, corresponding to a well-developed epithelium, secretory only in virgin queens, and glands of class III. On the other hand, mandibular glands have been found to be well-developed in workers and queens and they also have high amounts of mitochondria and smooth endoplasmic reticulum. Together, these data show that the mandibular and intramandibular glands of M. scutellaris virgin queens have morphological features that indicate the synthesis of compounds that are lipid in nature. Furthermore, both glands have a similar developmental degree in newly emerged and 7-day-old ones.
head glands /Melipona / communication / behavior / virgin queen
1. INTRODUCTION
Maintenance of insect societies is associated with chemical signals released by the exocrine glands, which are distributed in great numbers and diversity throughout the insect body (Cruz-Landim2009). The secretion of exocrine glands can be associated with recognition of individuals of the same colony through chem-ical signals, such as those reported in Apis mellifera capensis and Melipona scutellaris (Kerr et al. 2004; Wossler et al. 2006). With
regard to nestmate recognition, some stingless bees recognize virgin queens when they are a few days old, but these virgin queens are commonly killed by the workers (Kleinert and Imperatriz-Fonseca 1994; Imperatriz-Fonseca and Zucchi1995; Jarau et al.2009).
In bees, the exocrine glands are ectodermic in origin and can be classified into different classes according to the secretory apparatus. The exocrine glands of class I release substances that cross the body cuticle via the cuticular pore, which is a result of cuticle deposition after molting. Class III glands have two cell types: secretory cells and cells that form the conducting canal (Noirot and Quennedey
1991; Abdalla and Cruz-Landim2002). Among the exocrine glands of bees, the mandibular, Corresponding author: J. Serrão, jeserrao@ufv.br
Manuscript editor: James Nieh DOI:10.1007/s13592-014-0301-6
labial, and hypopharyngeal glands form the salivary gland system, which is present in queens, males, and workers (Cruz-Landim1967).
The mandibular glands of bees have a bag-shaped reservoir and secretory cells of class III (Nedel 1960). The mandibular glands may act as alarm and defense pheromones (Smith and Roubik 1983; van Zweden et al. 2011; Cruz-Lopez et al. 2005,2007; Schorkopf 2009) and may also participate in a queen’s attractiveness to worker bees in mated A. mellifera queens (Gary 1961).
The intramandibular glands are epidermal glands that differentiate during metamorphosis (Martins et al.2013) and this probably occurs in both sexes of all bee species, with two types of glandular cells inside the mandible (Abdalla and Cruz-Landim,2002). The first type is present in the region just below the epidermis and consists of cells of class III (Nedel 1960). The second type is class I formed by a secretory epidermis (Cruz-Landim 1967). However, the chemical nature and function of the intramandibular gland compounds are not known (Cruz-Landim et al.
2011).
In the stingless bees Melipona marginata, Melipona quadrifasciata, and Melipona beecheii, the virgin queens, a few days old, are recognized by workers, who may attack them (Kleinert and Imperatriz-Fonseca 1994; Imperatriz-Fonseca and Zucchi 1995; Jarau et al. 2009). In this sense, the behavior of the virgin queens may stimulate the workers and the most active queens are persecuted and attacked by the workers (Jarau et al. 2009). However, researches involving A. mellifera capensis and M. scutellaris suggest that recognition of indi-viduals of the same colony is through chemical signals (Kerr et al. 2004; Wossler et al. 2006). The possible function of the mandibular and intramandibular glands in virgin queens of stingless bees, of different ages, may be obtained by comparing the morphology of these glands in these queens, likely reported for workers in the stingless bee M. quadrifasciata (Cruz-Landim et al.2011).
The secretory activity and chemical compo-sition of the secretions from the glands may be
inferred indirectly on the basis of their cell morphology (Abdalla and Cruz-Landim, 2002). In this sense, some glands in the bee body show variations in size, morphology, and function according to caste, age, sex, and colony condi-tions (Costa-Leonardo 1981; Cruz-Landim
1994; Cruz-Landim 2009). The ultrastructure of the glands in A. mellifera queens, for example, has been reported to change according to the pheromone activity, as the queen ages (Dehazan et al. 1989). Similar age-related changes in the cell morphology and secretory activity of the mandibular glands have also been shown to occur in honeybee drones (Lensky et al. 1985).
The function of bee’s glands may be inferred from their morphology. Thus, the objective of this study is to compare the morphology of mandibular and intramandibular glands in virgin queens of different ages and in nurse and forager bees of M. scutellaris. We conducted assays to detect proteins and lipids in these glands to gain a better understanding of possible gland function. In virgin queens, we hypothe-size that these glands may be releasing different compounds that allow workers to recognize virgin queens.
2. MATERIALS AND METHODS 2.1. Animals
with pollen and honey from the storage pots of the same colonies, which was available ad libitum. This was a more viable method to collect virgin queens with known age, but this might differ from the natural conditions. Forager workers were collected when arriving at the nest entrances with corbiculae loaded with pollen, whereas the nurse workers were collect-ed inside the nest onto the brood combs.
2.2. Scanning electron microscopy
The mandibles and mandibular glands were dissected from two individuals and castes, from each nest, and transferred to Zamboni’s fixative solution (Stefanini et al. 1967), and dehydrated in a graded ethanol series. The samples were then transferred to hexamethyldisilazane for 5 min, air dried, and coated with gold (20 nm). The samples were examined using a scanning electron microscope (SEM) LEO VP1430.
2.3. Light microscopy
Twelve bees of each caste (3 from each colony), were cryoanesthetized for 1 min at −20 °C and decapitated shortly thereafter. The mandibles and mandibular glands were dissected and transferred to Zamboni’s fixative solution for 2 h. The samples were then dehydrated in a graded ethanol series and embedded in historesin. Slices, 4-μm thin, were stained with hematoxylin and eosin. Finally, ten slices from every bee were submitted for the following histochemical tests: periodic acid Schiff (PAS) to detect neutral polysaccharides and glycoconjugates; Nile blue to detect lipids; mercu-ry–bromophenol blue to detect total protein, accord-ing to Pearse (1985), and tests for acid and alkaline phosphatases according to van Noorden and Frederiks (1992).
2.4. Morphometry
Longitudinal histological sections of the entire length of the mandible and mandibular gland were randomly selected from ten bees of each caste, and prepared for light microscopy. Ten secretory cells (class III glands) that had an evident nucleus and cytoplasm were selected from each histological slice and measurements were taken of the total areas of the
cells and nuclei of these gland cells. Measurements were obtained with the aid of the software, Image Pro-Plus (Media Cybernetics).
2.5. Statistical analysis
The normal distribution of the morphometrical data (total cell and nucleus areas) were tested using the Kolmogorov-Smirnov test, and the data were subjected to one-way analysis of variance (ANOVA) followed by the multiple comparison Tukey’s test at a 5 % significance level.
2.6. Transmission electron microscopy Three bees of each caste were dissected and the mandibles and mandibular glands were removed and transferred to 2.5 % glutaraldehyde in a sodium cacodylate buffer of 0.1 M, pH 7.1, for 2 h. Following this, the samples were washed and post-fixed in 2 % osmium tetroxide in the same buffer for 2 h and dehydrated in 70 % ethanol. The samples were embedded in LR-White resin and, after poly-merization, were sectioned at 80–90 nm, stained with 1 % aqueous uranyl acetate and lead citrate, and analyzed in a transmission electron microscope (TEM), Zeiss EM 109. The contrast and brightness of the micrographs were improved using the soft-ware, Photoshop CS6.
3. RESULTS
3.1. Intramandibular glands
nucleus, but statistically showed a similar size (F = 0.842, p > 0.05; Figure 2a, b). The mandi-bles of the nurse and forager workers had pores on the inner surface, while in the queens the pores were concentrated at the outer surface of the mandible (Figure 3a–c).
In all the bees analyzed, there was a strong positive reaction for neutral polysac-charides in the excretory canal of the class III gland (Figure 4a, b). Only the nurse and forager workers showed a positive PAS reaction in the cytoplasm of the secretory cells. The histochemical test with mercury– bromophenol blue showed a positive result in the cytoplasm of secretory cells of all the bees analyzed, indicating the presence of
cytoplasmic proteins (Figure 4c). Lipids were also found in these glands (Figure 4d). There was no significant acid or alkaline phospha-t a s e a c t i v i t y i n t h e c e l l s o f t h e intramandibular glands of bees.
The ultrastructural analyses of the class III secretory cells in the intramandibular glands showed a well-developed, smooth endoplas-mic reticulum, as well as mitochondria, in all bees.
In addition to class III secretory cells, the virgin queens also had a developed secretory epithelium (class I gland), composed of columnar cells with spherical nuclei (Figure 1a), with a predominance of d e c o n d e n s e d c h r o m a t i n a n d a w e l l -Figure 1. Light micrographs of intramandibular
glands of Melipona scutellaris. a Newly emerged virgin queen showing the secretory epithelium (g1) well developed and the gland of class III (arrow). b Seven-day-old virgin queen showing gland of class III (g3) with irregular shape and nucleus (n) with
developed nucleolus, with the cytoplasm strongly PAS positive in the basal region (Figure 4a) and staining positively with mercury–bromophenol blue. Class I glands were not found along the entire length of the mandible, being located only at the epitheli-um lining of the external mandible surface. In workers, the mandible epidermis showed small cubic cells (Figure 1) with the nuclei containing condensed chromatin.
The cells of the class I intramandibular glands in virgin queens had nuclei with decondensed chromatin in the median cell region and a cytoplasm rich in smooth endoplasmic reticulum (Figure 5a, b). The basal portions of the cells had many plasma
membrane infoldings associated with mito-chondria, whereas in the cell apices, the plasma membrane had irregular projections forming an extensive labyrinth in the subcuticular space (Figure 5a). The ultra-structure of these cells was not analyzed in the workers, as the mandibular epithelium was nonglandular in these bees.
3.2. Mandibular glands
Mandibular glands with clustered class III cells were found in all castes of M. scutellaris. Moreover, there was a large reservoir with a thin wall of flattened cells and a thin membrane surrounding the secre-Figure 2. Morphometric data of intramandibular and
mandibular gland cells of Melipona scutellaris. a Total cell area of intramandibular class III gland showing difference between foragers and virgin queens (asterisk). b Nuclear area of intramandibular cell of class III gland without significant difference among castes. c Total cell area of mandibular gland
tory portion. The secretory cells of the mandibular glands were smaller in the forag-er workforag-ers (F =5.011, p< 0.05) than in both the newly emerged and 7-day-old virgin queens (Figure 2c, d).
The histochemical tests showed a strong positive reaction to neutral polysaccharides in the excretory canals, with the cytoplasm being weakly reactive in all the bees. Moreover, there was a strong reaction to protein in the cytoplasm of the cells of the mandibular glands of all the bees analyzed. Tests with Nile blue and acid and alkaline phosphatases were negative in the mandibular glands for all the bees.
The ultrastructural analyses showed that the cytoplasm of the mandibular glands was rich in smooth endoplasmic reticulum and mitochon-dria (Figure5c).
4. DISCUSSION
4.1. Intramandibular glands
We found two types of secretory cells in the intramandibular gland, in the mandibles of the virgin queens of M. scutellaris: class I and class III cells, according to the classification of Noirot and Quennedey (1991). Both classes of glands have been reported to occur in the mandibles of other bee species as well (Costa-Leonardo1978; Nedel1960; Santos et al.2009; Cruz-Landim et al.2011).
Intramandibular secretory cells of class III in newly emerged virgin queens differed morpholog-ically from those found in 7-day-old virgin queens, which were irregular in shape. This shrink-ing, which resulted in irregular gland cells, suggested that these gland cells suffered Figure 3. Scanning electronic micrographs of
man-dible surface of Melipona quadrifasciata. a Schemat-ic drawn showing a nomenclature of surface from of mandible of bee used in this paper. b Nurse worker
morphological age-related changes, but we did not find pyknotic nuclei, apoptotic bod-ies, or acid phosphatase in the cytoplasm, which were signs of advanced degenerative processes in insects (Armbruster et al. 1986; Dimitriadis and Kastritsis 1985; Skelton and Bowen1987; Jimenez and Gilliam1990; Gregorc and Bowen1997,1998; Teixeira et al.2013).
Despite the different morphology, the secre-tory cells in both ages of the virgin queens show a similar cellular area. This suggests that similar amounts of compounds are produced, as the area of the glandular cells is associated with the amount of secretion (Peters et al. 2010). These cells are thought to be responsible for producing lubricating compounds for the mandibles, and other substances related to the nest building tasks performed by the workers (Nedel 1960;
Santos et al. 2009). This hypothetical function of the intramandibular gland is supported by the large size of these cells in forager workers, who collect resins and other resources used in the nests of stingless bees (Michener 1974). How-ever, queens do not participate in any activities, other than reproduction. We have found lipid d r o p l e t s s t o r e d i n t h e c e l l s o f t h e intramandibular glands associated with a smooth endoplasmic reticulum, suggesting that the class III intramandibular glands play a role in lipid metabolism. Whether the secretions of these glands play a role in lubrication of the mouthparts or chemical communication remains an open question. Chemical signals in social insects, such as cuticular hydrocarbons, are lipid in nature, synthesized from the lipid stores in the glandular cells as well as in the complex Figure 4. Light micrographs of histochemical tests of
intramandibular glands of Melipona scutellaris. a Virgin queen 7-days old, showing strong positive reaction for glycogen (arrows) in cells of class III (g3) and in the basal portion of secretory epithelium (g1); PAS test. b Nurse worker evidencing
exocrine glands (Peeters et al. 1999; Sledge et al.2001; Mant et al.2005; Vasquez et al.2008; Hora et al.2010).
The hypertrophied, epithelial, secretory gland (class I) of the mandible of M. scutellaris virgin queens is rich in smooth endoplasmic reticulum. The occurrence of this hypertrophied epitheli-um, with a well-developed, smooth, endoplas-mic reticulum in virgin queens indicates the production of lipid compounds. However, in the stingless bees Plebeia emerina (Santos et al.
2 0 0 9) , M e l i p o n a r u f i v e n t r i s , a n d M . quadrifasciata anthidioides (Costa-Leonardo
1978), only the workers have a hypertrophied intramandibular epithelium, suggesting its pos-sible role in the manipulation of the adhesive resins and propolis in these insects (Santos et al.
2009).
In the basal portion of the cells of the hypertrophied mandibular epithelium of virgin queens, the plasma membrane has infoldings associated with mitochondria, whereas the cell apices have projections resulting in a labyrinth in the subcuticular space. Moreover, histo-chemical analysis shows PAS-positive areas, suggesting storage of glycogen, a molecule that may supply energy. This suggests that the epithelium participates in active transport of substances between the hemolymph and body surface. Transport of substances from the hemolymph to the body surface by the hypertrophied epithelium has been suggested to occur in ants (Serrão et al.2009; Hora et al.
2010).
T h e s i m u l t a n e o u s o c c u r r e n c e o f intramandibular glands of classes I and III in queens suggests that these two gland classes act independently in this caste. When different classes of glands are present together in one place and do not have a common reservoir, their secretions may differ in volatility and time of
secretion (Romani et al. 2003,2005; Marques-Silva et al. 2006).
Our findings show that the intramandibular glands of classes I and III have ultrastructural features of cells producing lipid compounds and the class III cells have a similar size in the virgin queens and nurse workers of M. scutellaris.
4.2. Mandibular gland
The mandibular glands of virgin queens of M. scutellaris, of different ages, showed secre-tory cells of similar sizes. This suggests that the mandibular glands of virgin queens maintain the same secretory activity at both ages. In the virgin queens of Scaptotrigona postica, the mandibular gland secretion is used as a form of communication with the males during the mating flight (Engels et al. 1997; Gracioli-Vitti et al.2004a).
The ultrastructure of the secretory cells of the mandibular glands of newly emerged and 7-day-old M. scutellaris virgin queens is quite similar. These cells have spherical nuclei with decondensed chromatin and prominent nucleoli, and a cytoplasm with abundant smooth endo-plasmic reticulum and mitochondria. These features indicate that the secretory cells of the mandibular glands of the M. scutellaris virgin queens synthesize compounds that are lipid in nature. In the social insects, like honeybees (Michener 1974), stingless bees (Gracioli-Vitti et al. 2004b), bumblebees (Cahliková et al.
2004), and the ant Polyergus rufescens (Grasso et al.2003), the secretions from the mandibular glands of queens are pheromones, which are hydrocarbons and esters synthesized from lipids (Blomquist and Howard, 2003). This contrasts with the findings for Melipona bicolor, in which the secretory cells of the mandibular glands of Figure 5.Transmission electron micrographs of
intramandibular and mandibular gland cells of virgin queens 7 days old of Melipona scutellaris. a Micrograph showing apical projections of the plasma membrane forming an extensive labyrinth in the subcuticular space (arrows) in intramandibular gland cell of class I. b
virgin queens show a rough endoplasmic retic-ulum and Golgi complexes (Gracioli et al.
2004), which are features of cells producing proteins other than lipids.
The secretory cells from the mandibular glands of the forager and nurse have a similar size and the forager worker has smaller man-dibular gland cells than the virgin queen, suggesting a decrease in compound production in forager bees. In stingless bees, the mandib-ular glands of workers are involved in the production of alarm and defense compounds (Smith and Roubik 1983; Hrncir et al. 2004; Cruz-Lopez et al.2005,2007; Jarau et al2006; Schorkopf et al.2007;2009; van Zweden et al.
2011).
Similar to those found in intramandibular glands, the mandibular ones have the same developmental degree in virgin queens and nurse workers, as also a possible role in lipid metabolism.
5. CONCLUSION
In conclusion, the intramandibular glands of class III of newly emerged virgin queens of M. scutellaris show morphological changes with aging of the queen. The intramandibular glands of class I occur only in virgin queens and they have some features of active transport of substances from the hemolymph to the body cuticle, whereas the mandibular glands of virgin queens may produce a higher amount of secretion than those in the forager workers. Overall, we have found that the intramandibular glands of classes I and III and the mandibular glands have a similar morphology and developmental degree in both virgin queens and workers, indicating that these glands may produce similar compounds between castes. Therefore, our prediction that intramandibular and mandibular glands may play a role in the recognition of the virgin queen by workers of M. quadrifasciata is not supported in this study.
ACKNOWLEDGMENTS
This research was supported by Brazilian Re-search Agencies National Council of ReRe-search
(CNPq) and Minas Gerais State Research Agency (FAPEMIG). Authors are grateful to Nucleus of Microscopy and Microanalysis (UFV) for technical assistance.
Morphologie des glandes mandibulaires et intramandibulaires chez les ouvrières et les reines vierges deMelipona scutellaris
Glandes céphaliques / stingless bee / communica-tion / comportement
Morphologie der Mandibeldrüsen und der Intramandibeldrüsen bei Arbeiterinnen und jungfräulichen Königinnen von Melipona scutellaris
Kopfdrüsen / Kommunikation / Verhalten / jungfräuliche Königin
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